Genomic instability is a cancer-enabling trait increasingly appreciated for its potential roles in cancer therapy. Cohesin is a highly-conserved molecular complex crucial to chromosomal stability, DNA replication, DNA repair, chromatin architecture, and transcriptional regulation. Cohesin is frequently somatically altered in cancer, with loss of its STAG2 component particularly common across myriad cancer types, including glioblastoma multiforme (GBM), Ewing sarcoma, melanoma, and urothelial cell carcinoma. GBM diagnosis carries a particularly poor prognosis. Despite this association, no strategy targeting cancer-associated cohesin defects is in clinical use. Nonetheless, negative genetic interactions, such as synthetic lethality (SL) and synthetic cytotoxicity (SC), involving STAG2 loss - once identified - could potentially be exploited to selectively kill cancer cells. SL/SC interactions have been found between cohesin components (including STAG2) and PARP-1, an enzyme important to DNA replication stress/damage response. However, the mechanism by which this sensitivity is conferred is poorly understood. Furthermore, cohesin and DNA replication/repair factors appear to be highly connected nodes in studies of negative genetic interactions to date, but the extent to which STAG2 loss in particular interacts with such factors is not understood. Moreover, genetic interactions between STAG2 and the remainder of the human genome have not been systematically explored. In the proposed research, we aim to clarify the relationships between cancer-associated cohesin alteration and (potential) cancer therapeutic targets. In Aim 1, the goal is to identify novel STAG2 SL/SC relationships. Whole genome CRISPR knockout library screens in STAG2-wild-type and isogenic STAG2-deficient GBM cell lines will be used to nominate new candidate SL interactions. SC interactions will be nominated using the same paired cell lines and screens with a subgenomic CRISPR knockout library targeting DNA replication/repair factors in the absence of and presence of standard-of-care therapeutics (which for GBM are temozolomide and ionizing radiation). Other candidate STAG2 negative interactions have been nominated through previous screens involving other cohesin components. Top candidates will each be subjected to validation and - if validated - further investigation. Aim 2 involves using GBM cell lines to decipher how PARP activity modulates cohesin complex composition, cellular levels, localization, and function in the presence and absence of STAG2. These studies will not only improve understanding of genome maintenance mechanisms and of known cohesin SL/SC relationships but also potentially uncover novel targets for cancer therapy. These findings, in turn, will hopefully be translated through pre-clinical and clinical studies in order to improve treatments for patients with cancer.